Recombinant DNA technology has made it possible to insert foreign DNA sequences into the genome of an organism, thus, altering the organism's phenotype. Early on it was recognized that transgenes integrated into a plant genome in a random fashion and in an unpredictable copy number. Thus, efforts were undertaken to control transgene integration in plants.
One method for inserting or modifying a DNA sequence involves introducing a transgenic DNA sequence flanked by sequences homologous to the genomic target and selecting or screening for a successful homologous recombination event. U.S. Pat. No. 5,527,695 issued to Hodges, et al., on Jun. 18, 1996 describes transforming eukaryotic cells with DNA sequences that are targeted to a predetermined sequence of the eukaryote's DNA. Specifically, the use of site-specific recombination is discussed. Transformed cells are identified through use of a selectable marker included part of the introduced DNA sequences.
Other methods relied on homologous recombination-based transgene integration as applied to prokaryotes and lower eukaryotes. With respect to plants, it was shown that artificially induced site-specific genomic double-stranded breaks in plant cells were repaired by homologous recombination with exogenously supplied DNA using two different pathways. (Puchta, et al., (1996) Proc Natl Acad Sci USA 93:5055-5060; US Patent Application Publication Number 2005/0172365A1 published Aug. 4, 2005; US Patent Application Publication Number 2006/0282914 published Dec. 14, 2006; WO 2005/028942 published Jun. 2, 2005).
Since the isolation, cloning, transfer and recombination of DNA segments, including coding sequences and non-coding sequences, is most conveniently carried out using restriction endonuclease enzymes. Much research has focused on studying and designing endonucleases such as WO 2004/067736 published Aug. 12, 2004; U.S. Pat. No. 5,792,632 issued to Dujon, et al., Aug. 11, 1998; U.S. Pat. No. 6,610,545 B2 issued to Dujon, et al., Aug. 26, 2003; Chevalier, et al., (2002) Mol Cell 10:895-905; Chevalier, et al., (2001) Nucleic Acids Res 29:3757-3774; Seligman, et al., (2002) Nucleic Acids Res 30:3870-3879.
Although a plethora of approaches have been developed to target a specific site for modification in the genome of a plant, there still remains a need for site-specific modification of a plant genome such that a fertile plant, having the alterated genome, can be recovered.